The long-term objective of this study is to develop and apply a new strategy for investigating molecular interactions that modulate eukaryotic gene expression in living cells and organisms. This new strategy combines the power of in vitro selection (SELEX) for isolating RNA aptamers that bind to active protein surfaces with high affinity, and new approaches for achieving rapid, high-level and controlled expression and activity of specific inhibitory aptamer RNAs (iaRNAs). The use of this strategy in higher eukaryotes provides a means of rapidly inactivating targeted domains of proteins, thereby allowing an assessment of their primary function and mechanism of action in vivo. The proposal has as its nexus the TATA Binding Protein (TBP), a central player in promoter structure and function. TBP's known physical interactions with a collection of cooperating and competing transcription factors are the targets of these inhibitors and the focus of these mechanistic studies. The specific aims are to: 1) Further characterize the structure and activity of selected high-affinity RNA aptamers against TBP (TBPaptamers) and use of these in vitro to address mechanistic roles of TBP-TATA interaction in transcription initiation and reinitiation. 2) Select and characterize in vitro additional aptamers that bind distinct surfaces of TBP and that bind to other transcription factors that interact with TBP. 3) Complete the design and test of expressions systems to study aptamers against the transcriptional machinery in vivo. 4) Use of aptamers to dissect the roles of TBP and its interacting factors in transcription in vivo. The selection and use of iaRNAs should find broad use in both basic research, as in the dissection of mechanisms of sophisticated macromolecular machines and regulatory circuits, and in therapeutic applications, as inhibitors of specific disease-causing proteins. Many diseases are known to be caused by either over-expression of an endogenous gene (such as an oncogene in cancer), or expression of exogenous genes (as in a virus infection). Even the general transcription factor TBP, a target of this study, is up-regulated in cancers and contributes to oncogenic transformation and tumor growth. The regulated in vivo expression of RNA aptamers that target disease-inducing proteins would prove to be of substantial therapeutic value.